Technical Abstract:
Current methods for detecting Cryptosporidium parvum (Cp) in environmental samples also detect species of Cryptosporidium that are not infectious for humans. The present study was undertaken to develop reagents for the specific identification of Cp oocysts. Antigens unique to Cp were identified by gradient SDS-PAGE/immunoblotting of oocyst protein from several different Cryptosporidium species. Antiserum was prepared against a unique 37 kDa Cp antigen and used to identify a recombinant DNA clone, designated rCp37. Cp oocyst expression of mRNA for Cp37 was confirmed by RT-PCR using primers derived from the rCp37 sequence. Antisera to purified rCp37 was prepared in rabbits and used to localize the respective native Cp37 antigen. Immunofluorescence studies showed that Cp37 was on the surface of Cp oocysts, but absent from C. baileyi. Immunoelectronmicroscopy assays showed that native Cp37 was distributed unevenly on the Cp oocyst surface. By SDS-PAGE/immunoblotting, anti-rCp37 sera recognized two native Cp antigens- Mr 37 and Mr 24. Purified rCp37 antigen was also used in an ELISA assay to differentiate immune from pre-immune sera. Studies are underway to test other Cryptosporidium species for Cp37 antigen and evaluate the usefulness of rCp37 for diagnosing cryptosporidiosis in humans and animals.